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Short answer
Polyethylenimine (PEI) transfection uses a cationic polymer to carry plasmid DNA into cultured mammalian cells, usually for transient expression of a gene of interest. It is a reagent-and-ratio method: results depend on the PEI type, its preparation and the mass ratio of DNA to PEI, which the Addgene protocol says must be determined empirically for each new batch.
Definition and scope
PEI is a synthetic polymer made mainly by ring-opening polymerization of aziridine (Ismail and Chou, 2025). In transfection it is the carrier, not a lipid: the polymer binds DNA and delivers it into the cell, where it is thought to promote endosomal escape through the proton sponge effect. Addgene describes its general method as transfecting mammalian cells using linear polyethylenimine, and states the outcome as transient expression of a gene of interest.
The search phrases people use for this topic name different things:
Polyethylenimine, PEI and “PEI polyethylenimine transfection”
These are the same polymer and the same method. The abbreviation PEI is used interchangeably with the full name.
Polyethylenimine transfection reagent
The “reagent” is the PEI preparation itself: a commercial product or a stock made in the lab. Addgene’s linear PEI protocol uses PEI of molecular weight 25,000 Da dissolved as a 1 mg/mL stock (100 mg of powder in 100 mL of deionized water, pH adjusted to 7.0, filtered through 0.22 µm).
Polyethylenimine transfection protocol
A protocol is a cell-line-specific recipe built on the reagent. This page explains the concepts behind one rather than repeating step lists; the Addgene “Transfection” protocol is the published reference for HEK293T cells, and CASRAI’s transfection methods, controls and biosafety guide places PEI among the other delivery methods.
How PEI transfection is used in practice
Addgene’s protocol gives a starting point of a 1:3 ratio of µg DNA to µg PEI, diluted in 500 µL of OptiPro SFM per 10 cm plate. In its sample data the 1:2 and 1:3 ratios gave high transfection efficiencies. The same page is explicit that the optimal ratio must be determined empirically for each new batch of 1 mg/mL PEI, and that there may be variation between batches of PEI depending on the user. Treat a published ratio as a starting range to test, not a fixed setting.
Two other inputs are called out by Addgene: different brands and lots of fetal bovine serum can promote or inhibit transfection, and endotoxins can inhibit transfection, so plasmid DNA purification should include an endotoxin removal step.
Linear versus branched PEI
PEI is produced in linear (LPEI) and branched (BPEI) forms depending on polymerization conditions and catalysts. The difference that Ismail and Chou state is in the amine groups:
| Form | Structure | Amine groups |
|---|---|---|
| Linear PEI (LPEI) | Straight chain | Secondary amines |
| Branched PEI (BPEI) | Branched | Primary, secondary and tertiary amines |
The Addgene protocol is written for linear PEI at 25,000 Da. When comparing a protocol with a product, check which form and molecular weight each one means before carrying ratios across.
Checklist: what varies between PEI transfections
Every item below is stated in the sources cited at the end; together they explain most reasons the same “PEI protocol” behaves differently in two labs.
- PEI form and molecular weight: linear or branched; the Addgene protocol specifies linear, 25,000 Da.
- Stock preparation: concentration (1 mg/mL in the Addgene protocol) and pH set to 7.0, noting that pH drifts quickly on acid or base addition.
- Stock storage: aliquots at -80 °C; a thawed stock kept at 4 °C for up to 2 months.
- Batch: re-determine the DNA to PEI mass ratio for each new batch of stock.
- Serum: FBS brand and lot can promote or inhibit transfection.
- Plasmid quality: endotoxin removal during DNA purification.
Where it matters for labs, clinics and supply decisions
CASRAI’s guide on transfection methods describes cationic polymers such as PEI as cheap and scalable and dominant in large-volume protein production. For labs that make PEI stock themselves, the checklist above is a record-keeping list: log the PEI form, molecular weight, stock date, pH and the ratio that was validated, so a later batch can be compared. Lipid-based alternatives are covered separately in lipofection, with a reagent-specific example in Lipofectamine 3000. Viral delivery is covered in lentiviral transduction, and physical delivery in the electroporator guide.
PEI is also cytotoxic, and Ismail and Chou identify cytotoxicity as a critical barrier to clinical translation, alongside non-degradability and serum instability. This is why the method is described here for cultured-cell work only.
Common misunderstandings
- PEI is a lipid reagent. It is not; it is a cationic polymer, so lipofection guidance does not transfer directly.
- One ratio works for every batch. Addgene says the ratio must be determined empirically for each new batch of PEI stock.
- PEI gives permanent expression. The Addgene method yields transient expression.
- Any PEI will do. Linear and branched PEI have different amine content, and a protocol is written for a stated form and molecular weight.
- If efficiency is low, the PEI is at fault. Serum lot and endotoxin in the plasmid prep are both documented inhibitors.
FAQ
What is polyethylenimine transfection?
It is the delivery of plasmid DNA into cultured mammalian cells using polyethylenimine, a cationic polymer, typically for transient expression of a gene of interest.
What is the difference between PEI and a PEI transfection reagent?
PEI is the polymer. The reagent is a particular preparation of it, either commercial or a lab-made stock such as the 1 mg/mL, pH 7.0, filter-sterilized stock described by Addgene.
What DNA to PEI ratio should I use?
Addgene’s linear PEI protocol starts at 1:3 (µg DNA to µg PEI) and reports high efficiency at 1:2 and 1:3, but states the optimum must be determined empirically for each new batch of PEI.
Is linear or branched PEI used for transfection?
The Addgene protocol uses linear PEI of 25,000 Da. Branched PEI contains primary, secondary and tertiary amines, whereas linear PEI contains secondary amines, so the two are not interchangeable in a protocol written for one.
Sources
- Addgene, “Transfection” protocol (linear PEI, HEK293T): addgene.org/protocols/transfection, checked 2026-10-29.
- Ismail A, Chou S-F. “Polyethylenimine Carriers for Drug and Gene Delivery.” Polymers (Basel) 2025;17(15):2150: PMC12349286, checked 2026-10-29.
